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1.
Ecotoxicol Environ Saf ; 183: 109508, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31408819

RESUMO

As a new type of antibacterial agent, nanosilver has attracted great attention in biomedical applications. However, the safety of nanosilver to humans and the environment has not been well elucidated. The objective of this study was to investigate the influence of nanosilver on novel effector mechanism of neutrophil extracellular traps (NETs), and its possible molecular mechanisms. In this study, nanosilver (10, 20 and 40 µg/mL) was incubated with neutrophils for 90 min. Then, nanosilver-induced the release of NETs was observed by laser confocal microscopy. Nanosilver-induced NETs release was also quantitatively detected by pico Green®. In addition, the role of NADPH oxidase, extracellular signal-regulated kinase (ERK) and p38 signaling pathways in nanosilver-induced NETs release were detected by the inhibitors and pico Green®. The results indicated that nanosilver significantly activated polymorphonuclear neutrophils (PMN) to release NETs, which was a DNA-based network structure modified with histones (H3) and neutrophil elastase (NE). The inhibitors of NADPH oxidase, ERK and p38 signaling pathways significantly inhibited the formation of nanosilver-induced NETs. Furthermore, nanosilver did not alter the extracellular lactate dehydrogenase (LDH) level of PMN cells. All these results showed that nanosilver significantly induced NETs release, and the potential molecular mechanisms were correlated with reactive oxygen species (ROS) production-dependent on NADPH oxidase, ERK and p38 signaling pathways, which might provide a new perspective on nanosilver-induced excess NETs release related to the host immune damage.


Assuntos
Antibacterianos/toxicidade , Armadilhas Extracelulares/metabolismo , Nanopartículas Metálicas/toxicidade , Neutrófilos/efeitos dos fármacos , Prata/toxicidade , Animais , Antibacterianos/química , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas Metálicas/química , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Prata/química
2.
Redox Biol ; 26: 101272, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31330481

RESUMO

BACKGROUND: NADPH oxidases (NOX) are a family of flavoenzymes that catalyze the formation of superoxide anion radical (O2•-) and/or hydrogen peroxide (H2O2). As major oxidant generators, NOX are associated with oxidative damage in numerous diseases and represent promising drug targets for several pathologies. Various small molecule NOX inhibitors are used in the literature, but their pharmacological characterization is often incomplete in terms of potency, specificity and mode of action. EXPERIMENTAL APPROACH: We used cell lines expressing high levels of human NOX isoforms (NOX1-5, DUOX1 and 2) to detect NOX-derived O2•- or H2O2 using a variety of specific probes. NOX inhibitory activity of diphenylene iodonium (DPI), apocynin, diapocynin, ebselen, GKT136901 and VAS2870 was tested on NOX isoforms in cellular and membrane assays. Additional assays were used to identify potential off target effects, such as antioxidant activity, interference with assays or acute cytotoxicity. KEY RESULTS: Cells expressing active NOX isoforms formed O2•-, except for DUOX1 and 2, and in all cases activation of NOX isoforms was associated with the detection of extracellular H2O2. Among all molecules tested, DPI elicited dose-dependent inhibition of all isoforms in all assays, however all other molecules tested displayed interesting pharmacological characteristics, but did not meet criteria for bona fide NOX inhibitors. CONCLUSION: Our findings indicate that experimental results obtained with widely used NOX inhibitors must be carefully interpreted and highlight the challenge of developing reliable pharmacological inhibitors of these key molecular targets.


Assuntos
Inibidores Enzimáticos/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Catálise , Linhagem Celular , Cromatografia Líquida , Descoberta de Drogas , Inibidores Enzimáticos/química , Humanos , Peróxido de Hidrogênio/metabolismo , Isoenzimas , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , NADPH Oxidases/genética , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem
3.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31249132

RESUMO

NADPH oxidases catalyze the production of reactive oxygen species and are involved in physio/pathological processes. NOX1 is highly expressed in colon cancer and promotes tumor growth. To investigate the efficacy of NOX1 inhibition as an anticancer strategy, tumors were grown in immunocompetent, immunodeficient, or NOX1-deficient mice and treated with the novel NOX1-selective inhibitor GKT771. GKT771 reduced tumor growth, lymph/angiogenesis, recruited proinflammatory macrophages, and natural killer T lymphocytes to the tumor microenvironment. GKT771 treatment was ineffective in immunodeficient mice bearing tumors regardless of their NOX-expressing status. Genetic ablation of host NOX1 also suppressed tumor growth. Combined treatment with the checkpoint inhibitor anti-PD1 antibody had a greater inhibitory effect on colon carcinoma growth than each compound alone. In conclusion, GKT771 suppressed tumor growth by inhibiting angiogenesis and enhancing the recruitment of immune cells. The antitumor activity of GKT771 requires an intact immune system and enhances anti-PD1 antibody activity. Based on these results, we propose blocking of NOX1 by GKT771 as a potential novel therapeutic strategy to treat colorectal cancer, particularly in combination with checkpoint inhibition.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , NADPH Oxidase 1/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Imunoterapia , Interferon gama/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Microambiente Tumoral/imunologia
4.
Exp Eye Res ; 185: 107692, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31189078

RESUMO

Many of the small molecule-based inhibitors of NADPH oxidase activity are largely inadequate to substantiate broad claims, often exhibiting a lack of Nox-isoform-specificity, and sometimes only acting as scavengers of reactive oxygen species (ROS). In the present study, we use a newly developed highly selective Nox4 inhibitor, GLX7013114, to modulate TGFß-induced lens epithelial to mesenchymal transition (EMT). Rat lens epithelial explants were pre-treated with 0.3  µM of GLX7013114, and then treated with 200 pg/ml of TGF-ß2 to induce lens EMT. ROS production was visualized microscopically using the superoxide fluorogenic probe, dihydroethidium (DHE). The EMT process was documented using phase-contrast microscopy, and molecular EMT markers were immunolabeled. qPCR was also performed to observe changes in EMT-associated genes. TGFß-induced ROS was evident at 8 h of culture and its intensity was found to be significantly reduced when GLX7013114 was applied, comparable to ROS levels measured in untreated explants. Using phase-contrast microscopy to follow TGFß-induced EMT over 5 days in the presence of the inhibitor, lens epithelial cells in explants became myofibroblastic by day 2 and underwent progressive apoptosis to reveal a bare lens capsule by day 5. Explants treated with TGFß and GLX7013114 had some increased cell survival; however, these differences were not significant. For the first time, Nox4 inhibition by GLX7013114 was shown to reduce the TGFß-induced gene expression of α-smooth muscle actin (αSMA), collagen 1a and fibronectin. GLX7013114, given that it appears to block aspects of TGFß-induced EMT, including ROS production, may be a new useful Nox4-selective inhibitor for further studies.


Assuntos
Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Cristalino/citologia , NADPH Oxidase 4/antagonistas & inibidores , Fator de Crescimento Transformador beta/farmacologia , Actinas/metabolismo , Animais , Colágeno Tipo I/metabolismo , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Microscopia de Contraste de Fase , NADPH Oxidases/antagonistas & inibidores , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
5.
Methods Mol Biol ; 1982: 75-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172467

RESUMO

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.


Assuntos
Ácido Araquidônico/metabolismo , NADPH Oxidases/metabolismo , Fagócitos/enzimologia , Ácido Araquidônico/química , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Colorimetria , Ativação Enzimática , Isomerismo , Estrutura Molecular , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/enzimologia , Fagócitos/imunologia , Proteínas Recombinantes de Fusão , Análise Espectral
6.
Methods Mol Biol ; 1982: 417-428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172486

RESUMO

A growing appreciation of NADPH oxidases (NOXs) as mediators of fundamental physiological processes and as important players in myriad diseases has led many laboratories on a search for specific inhibitors to help dissect the role in a given pathway or pathological condition. To date, there are only a few available inhibitors with a demonstrated specificity for a given isozyme. Among those, peptidic inhibitors have the advantage of being designed to target very specific protein-protein interactions that are essential for NOX activity. Herein, we provide the techniques to deliver these inhibitors both in cell culture as well as in vivo.


Assuntos
Desenho de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Proteínas de Transporte , Linhagem Celular , Células Cultivadas , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Humanos , Modelos Biológicos , Peptídeos/administração & dosagem , Ligação Proteica , Mapeamento de Interação de Proteínas , Espécies Reativas de Oxigênio/metabolismo
7.
Methods Mol Biol ; 1982: 429-446, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172487

RESUMO

Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , NADPH Oxidases/química , Trifosfato de Adenosina/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Interpretação Estatística de Dados , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas , Estrutura Molecular , NADPH Oxidases/antagonistas & inibidores , Oxirredução , Fenantridinas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Superóxidos/metabolismo
8.
Oxid Med Cell Longev ; 2019: 4578175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31210840

RESUMO

NADPH oxidase (Nox) is considered a major source of reactive oxygen species (ROS) in the heart in normal and pathological conditions. However, the role of Nox in severe acute pancreatitis- (SAP-) associated cardiac injury remains unclear. Therefore, we aim to investigate the contribution of Nox to SAP-associated cardiac injury and to explore the underlying molecular mechanisms. Apocynin, a Nox inhibitor, was given at 20 mg/kg for 30 min before SAP induction by a retrograde pancreatic duct injection of 5% sodium taurocholate. Histopathological staining, Nox activity and protein expression, oxidative stress markers, apoptosis and associated proteins, cardiac-related enzyme indexes, and cardiac function were assessed in the myocardium in SAP rats. The redox-sensitive MAPK signaling molecules were also examined by western blotting. SAP rats exhibited significant cardiac impairment along with increased Nox activity and protein expression, ROS production, cell apoptosis, and proapoptotic Bax and cleaved caspase-3 protein levels. Notably, Nox inhibition with apocynin prevented SAP-associated cardiac injury evidenced by a decreased histopathologic score, cardiac-related enzymes, and cardiac function through the reduction of ROS production and cell apoptosis. This protective role was further confirmed by a simulation experiment in vitro. Moreover, we found that SAP-induced activation in MAPK signaling molecules in cardiomyocytes was significantly attenuated by Nox inhibition. Our data provide the first evidence that Nox hyperactivation acts as the main source of ROS production in the myocardium, increases oxidative stress, and promotes cell apoptosis via activating the MAPK pathway, which ultimately results in cardiac injury in SAP.


Assuntos
Apoptose , Regulação Enzimológica da Expressão Gênica , Cardiopatias/enzimologia , Miocárdio/enzimologia , NADPH Oxidases/metabolismo , Pancreatite/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Caspase 3/biossíntese , Modelos Animais de Doenças , Cardiopatias/etiologia , Cardiopatias/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Miocárdio/patologia , NADPH Oxidases/antagonistas & inibidores , Pancreatite/induzido quimicamente , Pancreatite/complicações , Pancreatite/patologia , Ratos , Índice de Gravidade de Doença , Proteína X Associada a bcl-2/biossíntese
9.
Pharmacology ; 104(1-2): 36-42, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31055581

RESUMO

BACKGROUND: Endothelium-dependent dilatation is a predictor for vascular function. NADPH oxidase-derived O2- can inactivate nitric oxide and induce vascular injury. METHOD: The crude ethanolic extract of Lysimachia christinae Hance were separated out 4 fractions of different olarities by petroleum ether, ethyl acetate, n-butanol (NB), and aqueous. The endothelial integrity was appraised by vascular tension measurement. Dihydroethidium was utilized to observe the vascular reactive oxygen species (ROS) production. Western-blot was adopted to detect protein expression. RESULTS: Among the 4 fractions of L. christinae Hance, the NB fraction showed the most potent capacity of promoting endothelium-dependent vascular relaxation and inhibiting ROS formation in aortic rings, which were likely attributed by suppressing the expression of NAD(P)H oxidase subunit (gp91phox, p47phox, and p67phox) and enhancing the phosphorylation of endothelial NOS in vascular tone. CONCLUSIONS: These results suggest that the NB fraction possess the strongest vascular pharmacological activities among the crude ethanolic extract of L. christinae Hance, which may help us for purifying bioactive constituents and discovering new drugs from this herb in future.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Primulaceae/química , Vasodilatação/efeitos dos fármacos , 1-Butanol/química , Animais , Aorta Torácica , Fracionamento Químico/métodos , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/metabolismo , Etanol/química , Masculino , Camundongos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo
10.
Planta Med ; 85(9-10): 708-718, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30822814

RESUMO

Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O2 for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O2 for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H2O2 and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Hemeproteínas/metabolismo , Hipertensão Pulmonar/fisiopatologia , NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Salvia miltiorrhiza/química , Remodelação Ventricular/efeitos dos fármacos , Animais , Fator Natriurético Atrial/genética , Benzoxazóis/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/isolamento & purificação , Hemeproteínas/antagonistas & inibidores , Hipertensão Pulmonar/metabolismo , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidases/antagonistas & inibidores , Peptídeo Natriurético Encefálico/genética , Peroxidases/antagonistas & inibidores , Ratos Sprague-Dawley , Triazóis/farmacologia
11.
Redox Biol ; 22: 101134, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30798073

RESUMO

The activation of NADPH oxidase contributes to dopaminergic neurodegeneration and motor deficits in Parkinson's disease (PD). However, whether NADPH oxidase is involved in non-motor symptoms, especially cognitive dysfunction in PD remains unknown. This study is undertaken to characterize the effects of inhibition of NADPH oxidase by a widely used NADPH oxidase inhibitor apocynin on learning and memory deficits in paraquat and maneb-induced mouse PD model. Results showed that mice injected with paraquat and maneb displayed impairments of spatial learning and memory, which was associated with reduced tyrosine hydroxylase expression as well as increased neurodegeneration, synaptic loss, α-synuclein expression and Ser129-phosphorylation in the hippocampus. Interestingly, apocynin treatment significantly ameliorated learning and memory deficits as well as hippocampal neurodegeneration and α-synuclein pathology in mice treated with these two pesticides. Mechanistically, we found that apocynin mitigated paraquat and maneb-induced NADPH oxidase activation and related oxidative stress. Furthermore, reduced microglial activation and M1 polarization were observed in apocynin and paraquat and maneb co-treated mice compared with paraquat and maneb alone group. Finally, apocynin inhibited the activation of signal transducers and activators of transcription 1 (STAT1) and nuclear factor kappa B (NF-κB) pathways, two key regulatory factors for microglial M1 inflammatory responses, in paraquat and maneb-treated mice. Altogether, our findings implied that NADPH oxidase mediates learning and memory deficits in PD, and inhibition of NADPH oxidase by apocynin blocks impairments of learning and memory via the suppression of oxidative stress and neuroinflammation.


Assuntos
Acetofenonas/farmacologia , Inibidores Enzimáticos/farmacologia , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Animais , Comportamento Animal , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/metabolismo
12.
Int J Mol Sci ; 20(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717240

RESUMO

Three kinds of new water-soluble polysaccharides (FA, FB and FC) were isolated from wild mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam by the classical method "water extraction and alcohol precipitation" and purified by column chromatography. The Mw of FA, FB and FC ranged from 5690 Da to 38,340 Da. The three polysaccharide fractions in the fruiting body were mainly composed of 4 kinds of monosaccharides, including glucose, galactose, mannose, and arabinose, among which glucose and galactose were the major monosaccharides. The FTIR and NMR spectroscopy indicated that the skeleton of three fractions composed of a (1→4)-α-D-glycosidic backbone containing α-D-mannopyranose. In vitro anti-hypoxia activity data showed that three polysaccharide fractions possessed a significant effect on inhibiting PASM cells apoptosis under hypoxia. Among them, FC at the concentration of 200 µg/mL revealed a significant anti-hypoxia effect. These results revealed that the intracellular polysaccharides possessed potent anti-hypoxic activity, which might be related to inhibiting LDH and NADPH oxidase expression and promoting the formation of 5-hydroxytryptamine, dopamine, endothelins, acetylcholine. More importantly, FC showed good performance inducing KV1.5 expression and prohibiting KIR6.2 formation at protein level.


Assuntos
Agaricus/química , Polissacarídeos Fúngicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Arabinose/química , Sequência de Carboidratos , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fracionamento Químico/métodos , Carpóforos/química , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/isolamento & purificação , Galactose/química , Glucose/química , Humanos , Canal de Potássio Kv1.5/agonistas , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Manose/química , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oxigênio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo
13.
Cell Biol Int ; 43(5): 466-475, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30761659

RESUMO

Reactive oxygen species (ROS) are produced by NADPH oxidase (NOX), an enzyme that reduces oxygen by using NADPH as a substrate. Apocynin (APO) is a catechol that is used as a NOX inhibitor, and N-acetyl-cysteine ​​(NAC) can reduce intracellular ROS levels. In this work, the effect of APO and NAC on osteoclast formation were evaluated. APO and NAC significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive cells and the osteoclast area. We analyzed bone-marrow derived monocyte-macrophages (BMMs) that differentiated into osteoclasts after RANKL stimulation. Stimulation was associated with either APO or NAC treatment and osteoclastogenesis marker expression, including NFATc1, MMP-9, and DC-STAMP, was evaluated. APO decreased the intracellular calcium concentration by calcium channels other than ITPR1 and TPC2. On the other hand, APO reduced Tnfrsf11a (RANK) expression and did not alter Fam102a (EEIG1) expression. Therefore, our results demonstrate that APO inhibits osteoclastogenesis by the RANK-RANKL-related signaling pathways, decreases osteoclast markers, and reduces intracellular calcium concentration.


Assuntos
Acetofenonas/farmacologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Acetofenonas/metabolismo , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinase 9 da Matriz , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Fatores de Transcrição NFATC , Proteínas do Tecido Nervoso , Osteoclastos/metabolismo , Osteogênese/fisiologia , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/metabolismo
14.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1332-1340, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30763641

RESUMO

Diabetic nephropathy (DN) is one of the major long-term complications of diabetes. Lysophosphatidic acid (LPA) signaling has been implicated in renal fibrosis. In our previous study, we found that the LPA receptor 1/3 (LPAR1/3) antagonist, ki16425, protected against DN in diabetic db/db mice. Here, we investigated the effects of a specific pharmacological inhibitor of LPA receptor 1 (LPA1), AM095, on DN in streptozotocin (STZ)-induced diabetic mice to exclude a possible contribution of LPAR3 inhibition. AM095 treatment significantly reduced albuminuria and the albumin to creatinine ratio and significantly decreased the glomerular volume and tuft area in the treated group compared with the STZ-vehicle group. In the kidney of STZ-induced diabetic mice, the expression of LPAR1 mRNA and protein was positively correlated with oxidative stress. AM095 treatment inhibited LPA-induced reactive oxygen species production and NADPH oxidase expression as well as LPA-induced toll like receptor 4 (TLR4) expression in mesangial cells and in the kidney of STZ-induced diabetic mice. In addition, AM095 treatment suppressed LPA-induced pro-inflammatory cytokines and fibrotic factors expression through downregulation of phosphorylated NFκBp65 and c-Jun N-terminal kinases (JNK) in vitro and in the kidney of STZ-induced diabetic mice. Pharmacological or siRNA inhibition of TLR4 and NADPH oxidase mimicked the effects of AM095 in vitro. In conclusion, AM095 is effective in preventing the pathogenesis of DN by inhibiting TLR4/NF-κB and the NADPH oxidase system, consequently inhibiting the inflammatory signaling cascade in renal tissue of diabetic mice, suggesting that LPAR1 antagonism might provide a potential therapeutic target for DN.


Assuntos
Antioxidantes/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Hipoglicemiantes/farmacologia , Fenilacetatos/farmacologia , Receptores de Ácidos Lisofosfatídicos/genética , Receptor 4 Toll-Like/genética , Fator de Transcrição RelA/genética , Albuminúria/induzido quimicamente , Albuminúria/tratamento farmacológico , Albuminúria/genética , Albuminúria/metabolismo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Lisofosfolipídeos/antagonistas & inibidores , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Estreptozocina , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo
15.
Int J Mol Sci ; 20(2)2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650577

RESUMO

Gut microbiota are emerging as potential contributors to the regulation of host homeostasis. Dysbiosis of the gut microbiota associated with increased intestinal permeability facilitates the passage of endotoxins and other microbial products, including indoxyl sulfate in the circulation. Although an emerging body of evidence has suggested that indoxyl sulfate is a key substance for the development of chronic kidney disease, few studies have investigated the direct association of indoxyl sulfate with vascular function. We hypothesized that indoxyl sulfate adversely affects vascular function. Aortas isolated from male Wistar rat were examined in the presence or absence of indoxyl sulfate to assess the vascular function, including vasorelaxation and vasocontraction. Indoxyl sulfate (vs. vehicle) (1) decreased vasorelaxation induced by acetylcholine (ACh) but not by sodium nitroprusside; (2) had no significant alterations of noradrenaline-induced vasocontraction in the absence and presence of endothelium; (3) decreased adenylyl cyclase activator (forskolin)-induced vasorelaxation, while such a difference was eliminated by endothelial denudation; and (4) decreased vasorelaxations induced by calcium ionophore (A23187) and transient receptor potential vanilloid 4 agonist (GSK1016790A). The indoxyl sulfate-induced decrease in the vasorelaxations induced by ACh and A23187 increased by cell-permeant superoxide dismutase or by organic anion transporter inhibitor. However, apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effects on vasorelaxations induced by ACh, A23187, forskolin, and GSK1016790A in the presence of indoxyl sulfate. These results suggest that indoxyl sulfate directly affects the vascular function, particularly, endothelium-dependent vasorelaxation, and this effect may be attributable to increased oxidative stress after cell transportion via organic anion transporter, and such increased oxidative stress may not be attributable to activation of NADPH oxidase activation.


Assuntos
Aorta/patologia , Aorta/fisiopatologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Indicã/toxicidade , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Aorta/efeitos dos fármacos , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Colforsina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Nitroprussiato/farmacologia , Norepinefrina/farmacologia , Potássio/farmacologia , Ratos Wistar , Sulfonamidas/farmacologia , Superóxido Dismutase/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo , Vasoconstrição/efeitos dos fármacos
16.
ScientificWorldJournal ; 2019: 4639165, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30692874

RESUMO

We have identified ras guanyl releasing protein 2 (rasgrp2) as a blood vessel related gene from Xenopus embryo. In addition, we reported that RASGRP2 is also expressed in human umbilical vein endothelial cells (HUVEC). It is known that RASGRP2 activates Ras-related protein 1 (Rap1). However, the function of RASGRP2 in human vascular endothelium remains unknown. Therefore, we performed functional analysis of RASGRP2 using immortalized HUVEC (TERT HUVEC). We established a stable RASGRP2 overexpressing cell line (TERT HUVEC R) and mock cell line (mock). Furthermore, we compared the activity of Rap1 and the generation of intracellular reactive oxygen species (ROS), which is related to cell death, in both cell lines. Significant increase in Rap1 activity was observed in the TERT HUVEC R compared to the mock. Furthermore, apoptosis by tumor necrosis factor-α (TNF-α) stimulation was significantly more reduced in the TERT HUVEC R than in the mock. In the mock, apoptosis induced by TNF-α stimulation was decreased by pretreatment with diphenyleneiodonium (DPI), which is an inhibitor of NADPH oxidase (NOX). However, in the TERT HUVEC R, apoptosis induced by TNF-α stimulation was not reduced after pretreatment of DPI. Furthermore, there was no reduction in ROS production in the TERT HUVEC R after DPI pretreatment. In addition, the difference in the degree of apoptosis induced by TNF-α stimulation in both cell lines was consistent with the difference in ROS production in the cell lines. From these results, it was suggested that RASGRP2 activates Rap1 and the activated Rap1 suppresses apoptosis via NOX inhibition.


Assuntos
Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Endoteliais/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
17.
Int Immunopharmacol ; 68: 39-47, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611000

RESUMO

Acute lung injury (ALI) is one of the most serious complications in critically ill patients which often leads to morbidity and mortality. ALI characterized by severe inflammation of lungs occurs due to uncontrolled inflammatory immune response. However, the immunological mechanism(s) are far from being understood. The spleen tyrosine kinase (SYK), a key component of immune receptor signaling, plays a critical role in the modulation of inflammatory signaling in different immune cells. However, its role in ALI remains to be explored. Therefore, in this study, we investigated the effect of R406, a SYK inhibitor in lipopolysaccharide (LPS)-induced ALI mouse model. LPS led to increased SYK expression in neutrophils and gamma delta (γδ) T cells. This was associated with increased neutrophilic airway inflammation, vascular permeability, myeloperoxidase activity in the lung with upregulated expression of NADPH oxidase (NOX2)/MCP-1/TNF-α in neutrophils and IL-17A in γδ T cells/lung. Pulmonary inflammation was associated with higher mortality in mice with ALI. Inhibition of SYK signaling using R406 in the lung led to blockade of neutrophilic airway inflammation, vascular permeability, pro-inflammatory cytokine release and oxidative stress in innate immune cells, i.e. γδ T cells and neutrophils and the lung. R406 administered LPS group had better survival rate than LPS group. This suggests that SYK upregulation in γδ T cells and neutrophils plays an important role in inflammatory process during ALI. In conclusion, R406 exhibited a great potential to block the LPS-induced airway inflammation and mortality which could be developed as a potential future therapy in ALI.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Interleucina-17/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , Oxazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Quinase Syk/antagonistas & inibidores , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/imunologia , Animais , Anti-Inflamatórios/farmacologia , Linfócitos Intraepiteliais/efeitos dos fármacos , Linfócitos Intraepiteliais/imunologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Camundongos Endogâmicos BALB C , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Oxazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia
18.
Free Radic Biol Med ; 130: 140-150, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389498

RESUMO

The relative contributions of different mitochondrial and cytosolic sources of superoxide and hydrogen peroxide in cells are not well established because of a lack of suitable quantitative assays. To address this problem using resting C2C12 myoblasts we measured the effects of specific inhibitors that do not affect other pathways on the rate of appearance of hydrogen peroxide in the extracellular medium. We used inhibitors of NADPH oxidases (NOXs), suppressors of site IQ electron leak (S1QELs) at mitochondrial Complex I, and suppressors of site IIIQo electron leak (S3QELs) at mitochondrial Complex III. Around 40% of net cellular hydrogen peroxide release was from NOXs and approximately 45% was from the two mitochondrial sites; 30% from site IIIQo and 15% from site IQ. As expected, decreasing cytosolic antioxidant capacity by lowering glutathione levels increased the absolute rates from all sites without changing their proportions, whereas decreasing antioxidant defenses in the mitochondrial matrix increased only the absolute and relative contributions of the two mitochondrial sites. These results show directly that mitochondria are a major contributor to cytosolic hydrogen peroxide in resting C2C12 myoblasts, and provide the first direct evidence of superoxide/hydrogen peroxide production from site IQ in unstressed cells.


Assuntos
Citosol/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Mioblastos Esqueléticos/fisiologia , NADPH Oxidases/metabolismo , Animais , Domínio Catalítico , Células Cultivadas , Senescência Celular , Feminino , Camundongos , Camundongos Endogâmicos C3H , NADPH Oxidases/antagonistas & inibidores , Pirazóis/farmacologia , Piridonas/farmacologia , Ratos , Ratos Wistar
19.
Hum Cell ; 32(1): 22-30, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30386989

RESUMO

Cerebrovascular smooth muscle cells (SMCs) hyperplasia is an important contributor to cerebrovascular remodeling during hypertension. The aim of present study was to investigate the effects of Icariin on cerebrovascular SMCs proliferation and remodeling and the underlying mechanisms. The results revealed that Icariin administration attenuated the enhanced basilar artery constriction in angiotensin II (AngII)-induced hypertension rat model, as well as the inhibition of basilar artery diameter reduction in response to AngII and phenylephrine. In addition, histological analyses showed that Icariin also significantly ameliorated basilar artery remodeling in AngII hypertensive rats. In human brain vascular SMCs (HBVSMCs), AngII-induced cell proliferation, migration and invasion were markedly inhibited by Icariin treatment. Moreover, Icariin treatment largely limited AngII-induced the increase of reactive oxygen species (ROS) production in HBVSMCs, which was closely associated with cell proliferation. Analysis of the mechanisms showed that Icariin decreased ROS production via inhibiting NADPH oxidase activity but not mitochondria-derived ROS production. Further, Icariin promoted Nox2 degradation and consequently reduced its protein expression. In conclusion, these findings demonstrate that Icariin attenuates cerebrovascular SMCs hyperplasia and subsequent remodeling through inhibiting Nox2-containing NADPH oxidase activation, suggesting Icariin may be a potential therapeutic agent to prevent the onset and progression of stroke.


Assuntos
Angiotensina II/efeitos adversos , Encéfalo/irrigação sanguínea , Medicamentos de Ervas Chinesas , Flavonoides/farmacologia , Hipertensão/patologia , Hipertensão/fisiopatologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 2/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Fitoterapia , Remodelação Vascular/efeitos dos fármacos , Animais , Artéria Basilar/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Flavonoides/uso terapêutico , Humanos , Hiperplasia , Hipertensão/induzido quimicamente , Músculo Liso Vascular/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/prevenção & controle
20.
Ann Anat ; 222: 120-128, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30590121

RESUMO

Carthamus tinctorius L. (CT) has been widely used in Asian countries as a beverage and a folk medicine. The current study investigates the effect of CT extract on cardiac remodeling and possible mechanisms involved in Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats. Male Sprague-Dawley rats were administrated with L-NAME (40mg/kg/day) for five weeks to induce hypertension. Hypertensive rats were treated with CT extract (300mg/kg/day) or captopril (5mg/kg/day) or vehicle for a further two weeks. Treatment of hypertensive rats with CT extract or captopril significantly decreased systolic blood pressure, left ventricular (LV) hypertrophy and fibrosis, small intramyocardial coronary artery remodeling, and cardiac weight index. CT extract or captopril increased plasma nitric oxide metabolite (NOx) levels and reduced plasma transforming growth factor ß1 (TGF-ß1) level, together with downregulation of cardiac TGF-ß1 and matrix metalloproteinases-9 (MMP-9) expression. In addition, decreased plasma malondialdehyde (MDA) levels, consistent with downregulation of NADPH oxidase subunit gp91phox expression in heart tissue, was also observed after CT extract or captopril treatment. These findings suggest that CT extract alleviates cardiac remodeling in L-NAME-induced hypertensive rats, which is possibly related to inhibition of the NADPH oxidase-mediated TGF-ß1-MMP-9 pathway.


Assuntos
Anti-Hipertensivos/uso terapêutico , Carthamus tinctorius/química , Inibidores Enzimáticos , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , NADPH Oxidases/antagonistas & inibidores , NG-Nitroarginina Metil Éster , Extratos Vegetais/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Remodelação Ventricular/efeitos dos fármacos , Animais , Pressão Sanguínea , Captopril/uso terapêutico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Masculino , Ratos , Ratos Sprague-Dawley
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